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Background:Human serum albumin (HSA), the most abundant protein in plasma, is a monomeric multi-domain macromolecule with at least nine binding sites for endogenous and exogenous ligands. HSA displays an extraordinary ligand binding capacity as a depot and carrier for many compounds including most acidic drugs. Consequently, HSA has the potential to influence the pharmacokinetics and pharmacodynamics of drugs.Objective:In this review, the structural determinants of drug binding to the multiple sites of HSA are analyzed and discussed in detail. Moreover, insight into the allosteric and competitive mechanisms underpinning drug recognition, delivery, and efficacy are analyzed and discussed.Conclusion:As several factors can modulate drug binding to HSA (e.g., concurrent administration of drugs competing for the same binding site, ligand binding to allosteric-coupled clefts, genetic inherited diseases, and post-translational modifications), ligand binding to HSA is relevant not only under physiological conditions, but also in the pharmacological therapy management.

Olive quick decline syndrome (OQDS) is a disorder associated with bacterial infections caused by Xylella fastidiosa subsp. pauca ST53 in olive trees. Metabolic profile changes occurring in infected olive trees are still poorly investigated, but have the potential to unravel reliable biomarkers to be exploited for early diagnosis of infections. In this study, an untargeted metabolomic method using high-performance liquid chromatography coupled to quadrupole-time-of-flight high-resolution mass spectrometry (HPLC-ESI-Q-TOF-MS) was used to detect differences in samples (leaves) from healthy (Ctrl) and infected (Xf) olive trees. Both unsupervised and supervised data analysis clearly differentiated the groups. Different metabolites have been identified as potential specific biomarkers, and their characterization strongly suggests that metabolism of flavonoids and long-chain fatty acids is perturbed in Xf samples. In particular, a decrease in the defence capabilities of the host after Xf infection is proposed because of a significant dysregulation of some metabolites belonging to flavonoid family. Moreover, oleic acid is confirmed as a putative diffusible signal factor (DSF). This study provides new insights into the host-pathogen interactions and confirms LC-HRMS-based metabolomics as a powerful approach for disease-associated biomarkers discovery in plants. Graphical abstract

Human serum albumin (HSA) is the most abundant protein in plasma, contributing actively to oncotic pressure maintenance and fluid distribution between body compartments. HSA acts as the main carrier of fatty acids, recognizes metal ions, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays esterase, enolase, glucuronidase, and peroxidase (pseudo)-enzymatic activities. HSA-based catalysis is physiologically relevant, affecting the metabolism of endogenous and exogenous compounds including proteins, lipids, cholesterol, reactive oxygen species (ROS), and drugs. Catalytic properties of HSA are modulated by allosteric effectors, competitive inhibitors, chemical modifications, pathological conditions, and aging. HSA displays anti-oxidant properties and is critical for plasma detoxification from toxic agents and for pro-drugs activation. The enzymatic properties of HSA can be also exploited by chemical industries as a scaffold to produce libraries of catalysts with improved proficiency and stereoselectivity for water decontamination from poisonous agents and environmental contaminants, in the so called “green chemistry” field. Here, an overview of the intrinsic and metal dependent (pseudo-)enzymatic properties of HSA is reported to highlight the roles played by this multifaced protein.

Purpose: Recently, it has been demonstrated that Raman spectroscopy is able to differentiate between healthy parathyroid tissues and parathyroid adenoma based on the basis of a specific molecular fingerprint. However, to our knowledge, no previous studies have been performed to evaluate the metabolic profile of parathyroid adenoma. Therefore, we designed a proof of concept study aimed to investigate the glucose/fatty acid metabolisms, in addition to the mitochondrial changes, in solitary parathyroid adenoma and in healthy parathyroid glands. Methods: Nine females with primary hyperparathyroidism due to a solitary parathyroid adenoma and formal surgical indication for parathyroidectomy have been enrolled. At the time of surgery, the removed specimens were immediately submitted unfixed and a tissue slice of about 0.5 cm in diameter was obtained from the nodular lesion. The expression of selected metabolic enzymes and proteins has been evaluated by western blot analysis, using human parathyroid whole tissue lysates as control. Results: Data obtained highlighted an increase, compared with the healthy group, of: (i) the glucose uptake by the GLUT-1 receptor and its phosphorylation by hexokinase II (HXKII); (ii) the expression of 3-phosphoglycerate dehydrogenase (3-PGDH) and glucose-6-phosphate dehydrogenase (G6PD); (iii) lipids biosynthesis; and (iv) cytochrome c expression. Conclusions: Our findings highlight for the first time the parathyroid adenoma metabolic hallmarks that could represent potential molecular targets usable for the development of new pharmacological treatments, allowing to reduce surgical parathyroidectomy.

C.difficile infection (CDI) is not a merely &ldquo, gut-confined&rdquo, disease as toxemia could drive the development of CDI-related extra-intestinal effects. These effects could explain the high CDI-associated mortality, not just justified by diarrhea and dehydration. Here, the extra-intestinal effects of toxin A (TcdA) and B (TcdB) produced by C. difficile have been studied in vivo using the zebrafish embryo model. Noteworthy, protective properties of human serum albumin (HSA) towards toxins-induced extra-intestinal effects were also addressed. Zebrafish embryos were treated with TcdA, TcdB and/or HSA at 24 h post-fertilization. Embryos were analyzed for 48 h after treatment to check vital signs and morphological changes. Markers related to cardio-vascular damage and inflammation were evaluated by Real-Time quantitative PCR and/or western blotting. Both toxins induced cardiovascular damage in zebrafish embryos by different mechanisms: (i) direct toxicity (i.e., pericardial edema, cardiac chambers enlargement, endothelial alteration), (ii) increased hormonal production and release (i.e., atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)), (iii) alteration of the vascular system through the increase of the vascular endothelial growth factor (VEGF-A) levels, as well as of its receptors, (iv) pro-inflammatory response through high cytokines production (i.e., CXCL8, IL1B, IL6 and TNF&alpha, ) and (v) cell-mediated damage due to the increase in neutrophils number. In addition to cardiovascular damage, we observe skin alteration and inflammation. Finally, our data indicate a protective effect of HSA toward the toxins induced extra-intestinal effects. Together, our findings can serve as a starting point for humans&rsquo, studies to substantiate and understand the extra-intestinal effects observed in CDI patients.

The O2-mediated oxidation of all-β-barrel ferrous nitrosylated nitrobindin from Arabidopsis thaliana (At-Nb(II)-NO), Mycobacterium tuberculosis (Mt-Nb(II)-NO), and Homo sapiens (Hs-Nb(II)-NO) to ferric derivative (At-Nb(III), Mt-Nb(III), and Hs-Nb(III), respectively) has been investigated at pH 7.0 and 20.0 °C. Unlike ferrous nitrosylated horse myoglobin, human serum heme-albumin and human hemoglobin, the process in Nb(II)-NO is mono-exponential and linearly dependent on the O2 concentration, displaying a bimolecular behavior, characterized by kon = (6.3 ± 0.8) × 103 M−1 s−1, (1.4 ± 0.2) × 103 M−1 s−1, and (3.9 ± 0.5) × 103 M−1 s−1 for At-Nb(II)-NO, Mt-Nb(II)-NO, and Hs-Nb(II)-NO, respectively. No intermediate is detected, indicating that the O2 reaction with Nb(II)-NO is the rate-limiting step and that the subsequent conversion of the heme-Fe(III)-N(O)OO− species (i.e., N-bound peroxynitrite to heme-Fe(III)) to heme-Fe(III) and NO3− is much faster. A similar mechanism can be invoked for ferrous nitrosylated human neuroglobin and rabbit hemopexin, in which the heme-Fe(III)-N(O)OO− species is formed as well, although the rate-limiting step seems represented by the reshaping of the six-coordinated heme-Fe(III) complex. Although At-Nb(II)-NO and Mt-Nb(II)-NO are partially (while Hs-Nb(II)-NO is almost completely) penta-coordinated, density functional theory (DFT) calculations rule out that the cleavage of the proximal heme-Fe-His bond in Nb(II)-NO is responsible for the more stable heme-Fe(III)-N(O)OO− species. Moreover, the oxidation of the penta-coordinated heme-Fe(II)-NO adduct does not depend on O2 binding at the proximal side of the metal center. These features may instead reflect the peculiarity of Nb folding and of the heme environment, with a reduced steric constraint for the formation of the heme-Fe(III)-N(O)OO− complex.

Herein, the novel synthesis, characterisation and further application of magnetic multi-walled carbon nanotubes modified with polyamidoamine (PAMAM) dendrimers (MMWCNTs-D-NH2) as highly sensitive and selective recognition element in sensor development for the electrochemical determination of As(III) in water matrices is presented. The synthetized nanomaterials were characterised by Fourier transform- infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and transmission electron microscopy (TEM) analysis. Thus, the glassy carbon electrode (GCE) was subjected to functionalisation with the MMWCNTs-D-NH2 to produce the sensor. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetric (CV) measurements were performed to characterise the surface of modified electrode. The electrochemical performances of the developed sensor towards different concentration of As(III) were studied by square wave anodic stripping voltammetric (SWASV) measurements. The sensor shown its high sensitivity and selectivity against possible interferents, such as Hg(II), Pb(II), Cd(II), Cu(II). The limit of detection (LOD) was found to be 0.46 µgL−1. The suitability of the proposed sensor in spiked water samples (drinking, tap and mineral water) is also discussed.

Acute promyelocytic leukemia (APL) is a hematological disease characterized by a balanced reciprocal translocation that leads to the synthesis of the oncogenic fusion protein PML-RAR&alpha, APL is mainly managed by a differentiation therapy based on the administration of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, therapy resistance, differentiation syndrome, and relapses require the development of new low-toxicity therapies based on the induction of blasts differentiation. In keeping with this, we reasoned that a better understanding of the molecular mechanisms pivotal for ATRA-driven differentiation could definitely bolster the identification of new therapeutic strategies in APL patients. We thus performed an in-depth high-throughput transcriptional profile analysis and metabolic characterization of a well-established APL experimental model based on NB4 cells that represent an unevaluable tool to dissect the complex mechanism associated with ATRA-induced granulocytic differentiation. Pathway-reconstruction analysis using genome-wide transcriptional data has allowed us to identify the activation/inhibition of several cancer signaling pathways (e.g., inflammation, immune cell response, DNA repair, and cell proliferation) and master regulators (e.g., transcription factors, epigenetic regulators, and ligand-dependent nuclear receptors). Furthermore, we provide evidence of the regulation of a considerable set of metabolic genes involved in cancer metabolic reprogramming. Consistently, we found that ATRA treatment of NB4 cells drives the activation of aerobic glycolysis pathway and the reduction of OXPHOS-dependent ATP production. Overall, this study represents an important resource in understanding the molecular &ldquo, portfolio&rdquo, pivotal for APL differentiation, which can be explored for developing new therapeutic strategies.

Metal-free and Cu(II)-lipophilic porphyrins [H2Pp and Cu(II)Pp] loaded on titanium dioxide in the anatase phase (TiO2) were prepared and used as a heterogeneous catalyst for the photoreduction of Cr(VI) to Cr(III) in aqueous suspensions under UV–Vis light irradiation. TiO2 impregnated with copper(II) porphyrin [TiO2@Cu(II)Pp] was the most effective in photocatalyst reduction of toxic chromate Cr(VI) to non-toxic chromium Cr(III). We further evaluated an experimental design with the scope of fast optimization of the process conditions related to the use of TiO2 or TiO2-porphyrin based photocatalysts. A full factorial design as a chemometric tool was successfully employed for screening the affecting factors involved in photoconversion catalysis, with the modification of TiO2 both with porphyrin H2Pp and Cu(II)Pp. The studied experimental factors were the catalyst amount, the concentration of Cr(VI) ions, and the pH of the medium. The performed multivariate approach was successfully used for fast fitting and better evaluation of significant factors affecting the experimental responses, with the advantage of reducing the number of available experiments. Thus, the stability of the optimized TiO2 embedded Cu(II)Pp was investigated, confirming the high reproducibility and suitability for environmental purposes.

The anticoagulant therapy is widely used to prevent and treat thromboembolic events. Until the last decade, vitamin K antagonists were the only available oral anticoagulants; recently, direct oral anticoagulants (DOACs) have been developed. Since 55% to 95% of DOACs are bound to plasma proteins, the in silico docking and ligand-binding properties of drugs apixaban, betrixaban, dabigatran, edoxaban, and rivaroxaban and of the prodrug dabigatran etexilate to human serum albumin (HSA), the most abundant plasma protein, have been investigated. DOACs bind to the fatty acid (FA) site 1 (FA1) of ligand-free HSA, whereas they bind to the FA8 and FA9 sites of heme-Fe(III)- and myristic acid-bound HSA. DOACs binding to the FA1 site of ligand-free HSA has been validated by competitive inhibition of heme-Fe(III) recognition. Values of the dissociation equilibrium constant for DOACs binding to the FA1 site (ie, calc KDOAC ) derived from in silico docking simulations (ranging between 1.2 × 10-8 M and 1.4 × 10-6 M) agree with those determined experimentally from competitive inhibition of heme-Fe(III) binding (ie, exp KDOAC ; ranging between 2.5 × 10-7 M and 2.2 × 10-6 M). In addition, this study highlights the inequivalence of rivaroxaban binding to mammalian serum albumin. Given the HSA concentration in vivo (~7.5 × 10-4 M), values of KDOAC here determined indicate that the formation of the HSA:DOACs complexes in the absence and presence of FAs and heme-Fe(III) may occur in vivo. Therefore, HSA appears to be an important determinant for DOACs transport.

Nitrobindins (Nbs), constituting a heme-protein family spanning from bacteria to Homo sapiens, display an all-β-barrel structural organization. Human Nb has been described as a domain of the nuclear protein named THAP4, whose physiological function is still unknown. We report the first evidence of the heme-Fe(III)-based detoxification of peroxynitrite by the all-β-barrel C-terminal Nb-like domain of THAP4. Ferric human Nb (Nb(III)) catalyzes the conversion of peroxynitrite to (Formula presented.) and impairs the nitration of free l-tyrosine. The rate of human Nb(III)-mediated scavenging of peroxynitrite is similar to those of all-α-helical horse heart and sperm whale myoglobin and human hemoglobin, generally taken as the prototypes of all-α-helical heme-proteins. The heme-Fe(III) reactivity of all-β-barrel human Nb(III) and all-α-helical prototypical heme-proteins possibly reflects the out-to-in-plane transition of the heme-Fe(III)-atom preceding peroxynitrite binding. Human Nb(III) not only catalyzes the detoxification of peroxynitrite but also binds NO, possibly representing a target of reactive nitrogen species.

In the present paper, the biological effects of three different naphthalene diimides (NDIs) G-quadruplex (G4) ligands (H-NDI-Tyr, H-NDI-NMe2, and tetra-NDI-NMe2) were comparatively evaluated to those exerted by RHPS4, a well-characterized telomeric G4-ligand, in an in vitro model of glioblastoma. Data indicated that NDIs were very effective in blocking cell proliferation at nanomolar concentrations, although displaying a lower specificity for telomere targeting compared to RHPS4. In addition, differently from RHPS4, NDIs failed to enhance the effect of ionizing radiation, thus suggesting that additional targets other than telomeres could be involved in the strong NDI-mediated anti-proliferative effects. In order to test telomeric off-target action of NDIs, a panel of genes involved in tumor progression, DNA repair, telomere maintenance, and cell-cycle regulation were evaluated at transcriptional and translational level. Specifically, the compounds were able to cause a marked reduction of TERT and BCL2 amounts as well as to favor the accumulation of proteins involved in cell cycle control. A detailed cytofluorimetric analysis of cell cycle progression by means of bromodeoxyuridine (BrdU) incorporation and staining of phospho-histone H3 indicated that NDIs greatly reduce the progression through S-phase and lead to G1 accumulation of BrdU-positive cells. Taken together, these data indicated that, besides effects on telomeres and oncogenes such as Tert and Bcl2, nanomolar concentrations of NDIs determined a sustained block of cell proliferation by slowing down cell cycle progression during S-phase. In conclusion, our data indicate that NDIs G4-ligands are powerful antiproliferative agents, which act through mechanisms that ultimately lead to altered cell-cycle control.

Introduction: It is common for postmenopausal women to receive a vitamin D supplementation prescription to assist in preventing future falls and to maintain bone health. However, the association between vitamin D supplementation and physical fitness components has not been studied in older women with diabetes. Objective: We examined the influence of 12 months of vitamin D supplementation on the components of physical fitness in postmenopausal women with type 2 diabetes mellitus (T2DM). Methods: Thirty-five postmenopausal women (62.48 ± 7.67 years, 154.6 ± 5.11 cm, 73.93 ± 15.43 kg, 31.13 ± 5.82 BMI) with a diagnosis of T2DM participated in this longitudinal study where participants were supplemented with 1000 IU/day of vitamin D over 12 months. Subjects performed fasting blood samples, anthropometric assessments, body composition, clinical exams, and physical tests at 6-month intervals (P0, P6, and P12). Results and Conclusion: Vitamin D supplementation alone was effective in postmenopausal women in increasing serum vitamin D levels, altering muscle strength levels, promoting improvements in muscle function, as well as preventing and controlling fragility caused by T2DM and aging.

Sitagliptin is a hypoglycaemic agent used to reduce blood sugar levels in patients with type 2 diabetes mellitus (T2DM). Real time monitoring of sitagliptin levels is crucial to prevent overdose, which might cause liver, kidney and pancreatic diseases. As an alternative solution, a sitagliptin voltammetric sensor was fabricated using artificial receptors called electroactive molecularly imprinted polymer nanoparticles (nanoMIPs). The nanoMIP tagged with a redox probe (ferrocene) combines both the recognition and reporting functions. Traditional electrochemical sensors determine the redox activity of an analyte. Thus, they are influenced by interfering molecules and the nature of the sample. These innovative nanoMIPs allow us to easily design and customise sensors, increase their sensitivity and minimise the cross reactivity in biological samples. The present technology replaces the traditional enzyme–mediator pairs used in traditional biosensors. The polymer composition was optimized “in silico” using docking and screening methods. Nanoparticles were synthesized via free radical polymerization and a solid phase method and then characterized by infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and dynamic light scattering (DLS). The specific sitagliptin nanoparticles were covalently immobilized on platinum electrodes via silane and carbodiimide chemistry. The determination of sitagliptin in human plasma by a nanoMIP sensor was assessed by differential pulse voltammetry (DPV). The sensor current response was directly related to the change in nanoMIP conformation triggered by the analyte. The optimisation of the sensor response was made by adjusting (i) the silane concentration, (ii) nanoMIP concentration, and (iii) immobilization time. The sensor measurements in plasma revealed high selectivity and a sensitivity of 32.5 ± 0.6 nA pM−1 towards sitagliptin, and the limit of detection of the fabricated sensor was found to be 0.06 pM. The sensor displayed a satisfactory performance for the determination of sitagliptin in spiked human plasma, demonstrating the potential of this technology for drug monitoring and clinical diagnosis.

The pathogenicity of group AStreptococcus(GAS) is mediated by direct bacterial invasivity and toxin-associated damage. Among the extracellular products, the exotoxin streptolysin O (SLO) is produced by almost all GAS strains. SLO is a pore forming toxin (PFT) hemolitically active and extremely toxicin vivo. Recent evidence suggests that human serum albumin (HSA), the most abundant protein in plasma, is a player in the innate immunity “orchestra.” We previously demonstrated that HSA acts as a physiological buffer, partially neutralizingClostridioides difficiletoxins that reach the bloodstream after being produced in the colon. Here, we report thein vitroandex vivocapability of HSA to neutralize the cytotoxic and hemolytic effects of SLO. HSA binds SLO with high affinity at a non-conventional site located in domain II, which was previously reported to interact also withC. difficiletoxins. HSA:SLO recognition protects HEp-2 and A549 cells from cytotoxic effects and cell membrane permeabilization induced by SLO. Moreover, HSA inhibits the SLO-dependent hemolytic effect in red blood cells isolated from healthy human donors. The recognition of SLO by HSA may have a significant protective role in human serum and sustains the emerging hypothesis that HSA is an important constituent of the innate immunity system.

Infections caused by metallo-β-lactamase (MBL)-producing bacteria are emerging and carry a significant impact on patients' outcome. MBL producers are spread worldwide, both in community and hospital setting, with increasingly reported epidemic clusters and the search for MBL inhibitors is an important topic for public health. MBLs are zinc-dependent enzymes whose functioning can be hampered by zinc chelators. We evaluated the potential of six zinc chelators (disulfiram, nitroxoline, 5-amino-8-hydroxyquinoline, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA], cyclam, and N,N,N',N'-tetrakis (2-pyridymethyl) ethylenediamine [TPEN]) in restoring carbapenem activity against MBL producers. Zinc chelators alone or in combination with meropenem against MBL-producing Klebsiella pneumoniae, Chryseobacterium indologenes, Elizabethkingia meningoseptica, and Stenotrophomonas maltophilia isolates were tested in vitro and in vivo (Galleria mellonella). In vitro experiments showed a synergistic activity between TPEN and meropenem toward all the strains. Nitroxoline alone retained activity against S. maltophilia, C. indologenes, and E. meningoseptica. In vivo experiments showed that TPEN or nitroxoline in combination with meropenem increased survival in larvae infected with E. meningoseptica, S. maltophilia, and K. pneumoniae. Based on our data, zinc chelators are potential carbapenem adjuvants molecules (restoring carbapenem activity) against MBL-sustained infections and could represent an interesting option for infections induced by these microorganisms.

Haptoglobin related protein (Hpr) is a component of the trypanosome lytic factor (TLF), a complex acting in the innate immune response against African trypanosomes. Like haptoglobin (Hp), Hpr binds hemoglobin (Hb) in the blood, but unlike Hp, Hpr does not bind the CD163 receptor. Moreover, unlike Hp, Hpr retains the N-terminal signal peptide that is required for the association with Apolipoprotein L-1 (ApoL-1), a component of the TLF complex. Here, the molecular model of human Hpr has been built based on the high sequence identity with human Hp (91%). The structural bases of Hpr:Hpr dimerization and Hpr recognition by Hb and Trypanosoma brucei brucei Hp receptor (TbHpHbR) have been analyzed in parallel with those of Hp:Hp, Hp:Hb, and TbHpHbR:Hp:Hb complexes. We show that the Cys33-Cys33 intermolecular disulfide bridge that stabilizes the Hp1:Hp1 complex is replaced by the Phe33, Pro34, and Phe48 hydrophobic core in the Hpr:Hpr dimer. Moreover, we show that the N-terminal peptide of Hpr participates in the stabilization of the Hpr:Hpr dimer. Thus, the N-terminal peptide seems to have been retained in Hpr to mediate its critical role in the human innate immunity towards Trypanosoma brucei brucei infection. Communicated by Ramaswamy H. Sarma

Background Glioblastoma is the most aggressive and most lethal primary brain tumor in the adulthood. Current standard therapies are not curative and novel therapeutic options are urgently required. Present knowledge suggests that the continued glioblastoma growth and recurrence is determined by glioblastoma stem-like cells (GSCs), which display self-renewal, tumorigenic potential, and increased radio- and chemo-resistance. The G-quadruplex ligand RHPS4 displays in vitro radiosensitizing effect in GBM radioresistant cells through the targeting and dysfunctionalization of telomeres but RHPS4 and Ionizing Radiation (IR) combined treatment efficacy in vivo has not been explored so far. Methods RHPS4 and IR combined effects were tested in vivo in a heterotopic mice xenograft model and in vitro in stem-like cells derived from U251MG and from four GBM patients. Cell growth assays, cytogenetic analysis, immunoblotting, gene expression and cytofluorimetric analysis were performed in order to characterize the response of differentiated and stem-like cells to RHPS4 and IR in single and combined treatments. Results RHPS4 administration and IR exposure is very effective in blocking tumor growth in vivo up to 65 days. The tumor volume reduction and the long-term tumor control suggested the targeting of the stem cell compartment. Interestingly, RHPS4 treatment was able to strongly reduce cell proliferation in GSCs but, unexpectedly, did not synergize with IR. Lack of radiosensitization was supported by the GSCs telomeric-resistance observed as the total absence of telomere-involving chromosomal aberrations. Remarkably, RHPS4 treatment determined a strong reduction of CHK1 and RAD51 proteins and transcript levels suggesting that the inhibition of GSCs growth is determined by the impairment of the replication stress (RS) response and DNA repair. Conclusions We propose that the potent antiproliferative effect of RHPS4 in GSCs is not determined by telomeric dysfunction but is achieved by the induction of RS and by the concomitant depletion of CHK1 and RAD51, leading to DNA damage and cell death. These data open to novel therapeutic options for the targeting of GSCs, indicating that the combined inhibition of cell-cycle checkpoints and DNA repair proteins provides the most effective means to overcome resistance of GSC to genotoxic insults. Electronic supplementary material The online version of this article (10.1186/s13046-019-1293-x) contains supplementary material, which is available to authorized users.

Chronic inflammation increases the risk of developing certain types of cancer, such as colorectal cancer (CRC). The oxidative metabolism of polyunsaturated fatty acids (PUFAs) has a strong effect on colonic tumorigenesis and the levels of arachidonic acid (AA) and eicosapentaenoic acid (EPA) can contribute to the development of an inflammatory microenvironment. Aim of this study was to evaluate the possible differences in the AA/EPA ratio tissue levels between CRC patients with and without synchronous metastases. Moreover, the expression of the most important inflammatory enzymes and mediators, linked with the AA/EPA ratio, have been also assessed. Sixty-eight patients with CRC were enrolled in the study, of which 33 patients with synchronous metastasis. Fatty acid profile analysis in tissue samples was done to examine the levels of AA and EPA. High levels of the AA/EPA ratio were detected in tumor tissue of patients with metastatic CRC. Moreover, an increase of expression of the main enzymes and mediators involved in inflammation was also detected in the same samples. The lipidomic approach of inflammation allows to evaluate lipid homeostasis changes that occur in cancer and in its metastatic process, in order to identify new biomarkers to be introduced into clinical practice. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

Proteases are a class of enzymes that lower the activation energy for the cleavage of the peptide bonds by polarizing the carbonyl group. The catalytic mechanism of proteases is characterized by the formation and the dissociation of a tetrahedral acyl-intermediate. The rate-limiting step in catalysis is either the acylation process (leading to the release of the newly formed $$ {-}{\text{NH}}_{3}^{ + } $$ terminal) or the subsequent deacylation step (leading to the release of the newly formed –COO− terminal). As a case, the detailed kinetic analysis for the hydrolysis of the chromogenic substrate Mu-His-Ser-Ser-Lys-Leu-Gln-AMC (wherein Mu is the morpholinocarbonyl protecting group and AMC is the 7-amino-4-methylcoumarin chromophoric group) by the prostate-specific antigen (PSA) is reported here. The pH dependence of the catalytic parameters clearly indicates the existence of protonation/deprotonation processes involving (at least) two ionizing groups in the proximity of the active site. In view of the physio-pathological relevance of PSA in prostate diseases (including cancer), the detailed analysis of the catalytic parameters opens new scenarios for the design of selective inhibitors, which might influence the “in vivo” activity of this protease.

In the present work, we evaluated the effects of a mixture of biocontrol agents against two toxigenic strains of Penicillium expansum isolated in Argentine Patagonia from pome fruits. The two strains, INTA-5 and INTA-10, were previusly selected among ten strains coming from the Alto Valle (Rio Negro-Argentina) for their high production of patulin. For the biocontrol, Kosakonia radicincitans, Cryptococcus laurentii, and Rhodosporidium fluviale were tested in vitro experiments on Potato Dextrose Agar (PDA) dishes against the INTA-5 and INTA-10 strains. The bacterium K. radicincitans and the yeast C. laurentii were selected to be used in a mixture due to their capacity to control the fungus and reduce the mycotoxin severely. In vitro assays with the mixture showed a high antagonism against P. expansum INTA-5 and INTA-10, at 21 d of incubation at 25 °C and a patulin reduction of 98%. The mixture of microorganisms was also effective in apples stored at 25 °C for 10 d and 4 °C for 30 d. At cold storage, the mixture controlled moderately the development of rot and decreased patulin concentration. At 25 °C, the pathogen's optimal growth temperature, the mixture of Biological Control Agent (BCAs) assured both the control of rot and decrease of patulin concentration. The combination of two microorganisms, with different requirements and abilities, resulted in a mix with a strong antagonism against P. expansum with the capability to decrease the patulin concentration. Treatment with the selected mixture could be a good option for controlling strains with different behaviours and in different environmental conditions.

Background: The pathogenic effects of Clostridium difficile are primarily attributable to the production of the large protein toxins (C difficile toxins [Tcd]) A (TcdA) and B (TcdB). These toxins monoglucosylate Rho GTPases in the cytosol of host cells, causing destruction of the actin cytoskeleton with cytotoxic effects. Low human serum albumin (HSA) levels indicate a higher risk of acquiring and developing a severe C difficile infection (CDI) and are associated with recurrent and fatal disease. Methods: We used a combined approach based on docking simulation and biochemical analyses that were performed in vitro on purified proteins and in human epithelial colorectal adenocarcinoma cells (Caco-2), and in vivo on stem cell-derived human intestinal organoids and zebrafish embryos. Results: Our results show that HSA specifically binds via its domain II to TcdA and TcdB and thereby induces their autoproteolytic cleavage at physiological concentrations. This process impairs toxin internalization into the host cells and reduces the toxin-dependent glucosylation of Rho proteins. Conclusions: Our data provide evidence for a specific HSA-dependent self-defense mechanism against C difficile toxins and provide an explanation for the clinical correlation between CDI severity and hypoalbuminemia.

Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H2O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural–functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

Herein we report the fabrication, characterisation and application of a novel voltammetric sensor based on an electrosynthesised ion imprinted polymer (IIP) film for the determination of copper(II) in water samples. This sensitive and highly selective sensor was prepared by electropolymerisation of p-phenylenediamine in the presence of Cu2+ as template, combined with the screen-printed technology for the transducer element. Electrochemical methods were used to control the electropolymerisation process, the template removal and the subsequent rebinding. The electrosynthesised film on the surface of screen-printed platinum electrodes was characterised by cyclic voltammetry (CV), scanning electron microscopy (SEM), and FTIR-ATR analysis. The incubation of the IIP-modified electrode with Cu2+ resulted in an increase of the redox properties of the obtained polymer. The performance of the proposed sensor was observed in acetate buffer (50 mM, pH = 5) over the Cu2+ concentration range 0.95–244 nM, with a limit of detection of 2.7 nM. The multivariate approach was employed in order to evaluate the influence of four variables on the sensitivities of the imprinted sensor towards the template. The IIP sensor showed high affinity to copper in comparison with the non-imprinted polymer (NIP). The reproducibility of the sensor was evaluated on five different sensors and it was estimated as 7.75 % (RSD). Selectivity studies were performed against several potential interferents, such as Ni2+, Mn2+, Ag+, Hg2+, Zn2+, Co2+, Pb2+ and Cd2+ ions. The application of the proposed sensor in spiked drinking water revealed a good recovery (95–105 %).

Haptoglobin (Hp) binds human hemoglobin (Hb), contributing to prevent extra-erythrocytic Hb-induced damage. Hp forms preferentially complexes with αβ dimers, displaying heme-based reactivity. Here, kinetics and thermodynamics of fluoride and azide binding to ferric human Hb (Hb(III)) complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively) are reported (pH 7.0 and 20.0 °C). Fluoride binds to Hp1-1:Hb(III) and Hp2-2:Hb(III) with a one-step kinetic and equilibrium behavior. In contrast, kinetics of azide binding to and dissociation from Hp1-1:Hb(III)(–N3−) and Hp2-2:Hb(III)(–N3−) follow a two-step process. However, azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) should reflect azide binding to the ferric β and α chains, respectively, as also proposed for the similar behavior observed in Hb(III). Present results highlight the ligand-dependent kinetic inequivalence of Hb subunits in the ferric form, reflecting structural differences between the two subunits in the interaction with some ferric ligands.

The endocannabinoid system is a unique neuromodulatory system that affects a wide range of biological processes and maintains the homeostasis in all mammal body systems. In recent years, several pharmacological tools to target endocannabinoid neurotransmission have been developed, including direct and indirect cannabinoid agonists and cannabinoid antagonists. Due to their hydrophobic nature, cannabinoid agonists and antagonists need to bind specific transporters to allow their distribution in body fluids. Human serum albumin (HSA), the most abundant plasma protein, is a key determinant of drug pharmacokinetics. As HSA binds both the endocannabinoid anandamide and the active ingredient of Cannabis sativa, Δ-9-tetrahydrocannabinol, we hypothesize that HSA can be the most important carrier of cannabinoid drugs. In silico docking observations strongly indicate that HSA avidly binds the indirect cannabinoid agonists URB597, AM5206, JZL184, JZL195, and AM404, the direct cannabinoid agonists WIN55,212-2 and CP55,940, and the prototypical cannabinoid antagonist/inverse agonist SR141716. Values of the free energy for cannabinoid drugs binding to HSA range between -5.4 kcal mol-1 and -10.9 kcal mol-1 . Accounting for the HSA concentration in vivo (∼ 7.5 × 10-4 M), values of the free energy here determined suggest that the formation of the HSA:cannabinoid drug complexes may occur in vivo. Therefore, HSA appears to be an important determinant for cannabinoid efficacy and may guide the choice of the drug dose regimen to optimize drug efficacy and to avoid drug-related toxicity. © 2017 IUBMB Life, 69(11):834-840, 2017.

Human serum albumin (HSA) represents an important determinant of plasma oncotic pressure and a relevant factor that modulates fluid distribution between the body compartments. Moreover, HSA (i) represents the depot and transporter of several compounds, both endogenous and exogenous, (ii) affects the pharmacokinetics of many drugs, (iii) regulates chemical modifications of some ligands, (iv) shows (pseudo-)enzymatic properties, (v) inactivates some toxic compounds, and (vi) displays anti-oxidant properties. HSA binding and (pseudo-)enzymatic properties are regulated competitively, allosterically, and by covalent modifications. While competitive inhibition of HSA binding properties is evident, allosteric mechanisms and covalent modifications affecting HSA reactivity are less clear. In several pathological conditions in which free heme-Fe levels increase, the buffering capacity of plasma hemopexin is overwhelmed and most of heme-Fe binds to the fatty acid site 1 of HSA. HSA-heme-Fe displays globin-like properties; in turn, heme-Fe modulates competitively and allosterically HSA binding and reactivity properties. Remarkably, heme-Fe-mediated HSA properties are time-dependent, representing a case for "chronosteric effects". Here, we review the drug-based modulation of (i) heme-Fe-recognition by HSA and (ii) heme-Fe-mediated reactivity.

Classical all α-helical globins are present in all living organisms and are ordered in three lineages: (i) flavohemoglobins and single domain globins, (ii) protoglobins and globin coupled sensors and (iii) truncated hemoglobins, displaying the 3/3 or the 2/2 all α-helical fold. However, over the last two decades, all β-barrel and mixed α-helical-β-barrel heme-proteins displaying heme-based functional properties (e.g. ligand binding, transport and sensing) closely similar to those of all α-helical globins have been reported. Monomeric nitrophorins (NPs) and α1-microglobulin (α1-m), belonging to the lipocalin superfamily and nitrobindins (Nbs) represent prototypical heme-proteins displaying the all β-barrel and mixed α-helical-β-barrel folds. NPs are confined to the Reduviidae and Cimicidae families of Heteroptera, whereas α1-m and Nbs constitute heme-protein families spanning bacteria toHomo sapiens. The structural organization and the reactivity of the stable ferric solvent-exposed heme-Fe atom suggest that NPs and Nbs are devoted to NO transport, storage and sensing, whereasHs-α1-m participates in heme metabolism. Here, the structural and functional properties of NPs and Nbs are reviewed in parallel with those of sperm whale myoglobin, which is generally taken as the prototype of monomeric globins.

A highly sensitive disposable electrochemical sensor for paracetamol was devised using electroactive molecularly imprinted polymers nanoparticles (nanoMIPs). NanoMIPs were prepared by solid-phase synthesis. Polymer composition included itaconic acid as a specific functional monomer and ferrocene as redox label, which confers electroactivity to the nanoparticles. NanoMIPs were characterised by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) and scanning electron microscopy (SEM). Sensors were fabricated by covalent attachment of nanoMIPs on screen-printed carbon electrodes and then employed for electrochemical determination of paracetamol using differential pulse voltammetry (DPV). The sensor was successfully evaluated in spiked human plasma with recoveries at 94–108 % and presenting a sensitivity of 6.18 ± 0.22 μA mM−1. The limit of detection and limit of quantification for the sensor were found to be 50 μM and 167 μM, respectively, in a linear concentration range between 0.1 and 1 mM. High selectivity was demonstrated, with no interference found in the presence of caffeine, procainamide or ethyl 4-aminobenzoate. The sensors exhibited high reproducibility (RSD, 4.8 %), fast response time (∼8 s) and acceptable shelf life (90 days), confirming its suitability for point of care diagnostic applications.

Background Infections caused by multidrug-resistant Enterobacteriaceae are hard to treat and life-threatening due to reduced therapeutic options. Systemic infections caused by Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae strains have increased in many European regions, becoming frequent in many clinical settings, and are associated with high mortality. The co-formulation of ceftazidime, a third-generation cephalosporin, with avibactam, a new suicide inhibitor beta-lactamase inhibitor able to block most Klebsiella pneumoniae carbapenemases, has been recently licensed, with promising results in patients with limited or absent therapeutic options. Little is known, however, as to the efficacy of such a combination in patients with soft tissue infections caused by multidrug-resistant Klebsiella pneumoniae carbapenemase-producing strains of Klebsiella pneumoniae. Case presentation A Caucasian 53-year-old man with paraplegia suffered multiple vertebral fractures due to a car crash. He was treated with external fixators that became infected early after insertion and were repeatedly and inefficiently treated with multiple antibiotics. He suffered repeated septic episodes caused by Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae strains with a multidrug-resistant profile. Meropenem, tigecycline, and colistin combinations allowed only temporary improvements, but septic shock episodes recurred, in spite of removal of infected external fixators. After approval of pre-marketing prescription by our local Ethics Committee, full clinical resolution was obtained with a compassionate treatment using meropenem and ceftazidime/avibactam in combination for 16 days. Conclusions Our experience provides additional evidence that ceftazidime/avibactam, possibly in combination with meropenem rescued by avibactam, may be an efficacious treatment option also for complicated skin and soft tissue infections caused by multidrug-resistant strains of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae.

Cellular damage occurring under oxidative conditions has been ascribed mainly to the formation of peroxynitrite (ONOOH/ONOO(-)) that originates from the reaction of NO(*) with O(2) (*-). The detrimental effects of peroxynitrite are exacerbated by the reaction with CO(2) that leads to ONOOC(O)O(-), which further decays to the strong oxidant radicals NO(2) (*) and CO(3) (*-). The reaction with CO(2), however, may redirect peroxynitrite specificity. An excessive formation of peroxynitrite represents an important mechanism contributing to the DNA damage, the inactivation of metabolic enzymes, ionic pumps, and structural proteins, and the disruption of cell membranes. Because of its ability to oxidize biomolecules, peroxynitrite is implicated in an increasing list of diseases, including neurodegenerative and cardiovascular disorders, inflammation, pain, autoimmunity, cancer, and aging. However, peroxynitrite displays also protective activities: (i) at high concentrations, it shows anti-viral, anti-microbial, and anti-parasitic actions; and (ii) at low concentrations, it stimulates protective mechanisms in the cardiovascular, nervous, and respiratory systems. The detrimental effects of peroxynitrite and related reactive species are impaired by (pseudo-) enzymatic systems, mainly represented by heme-proteins (e.g., hemoglobin and myoglobin). Here, we report biochemical aspects of peroxynitrite actions being at the root of its biomedical effects.

Fil: Cozzolino, Mariana Emilia. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Area Microbiologia; Argentina

Human serum albumin (HSA), the most abundant protein in plasma, displays several functions including heme transfer from high- and low-density lipoproteins to hemopexin; therefore, the HSA-heme complex acquires transiently heme-based (pseudo-)enzymatic properties. In particular, ferric human serum heme-albumin (HSA-heme) and ferrous nitrosylated HSA-heme inactivate peroxynitrite, and ferrous HSA-heme catalyzes the conversion of nitrite to nitrogen monoxide. The (pseudo-)enzymatic properties of HSA-heme are modulated allosterically by endogenous and exogenous ligands, such as drugs. The modulation of ligand binding to plasma proteins is relevant not only under physiological conditions but also in the pharmacological therapy management. Here, drug-dependent HSA-heme properties are reviewed from the functional and structural viewpoints. In particular, the drug-dependent five- to six-coordination transition of the heme-Fe atom is at the root of the allosteric modulation of the HSA-heme reactivity.

Lipocalins are an evolutionarily conserved family of proteins that bind and transport a variety of exogenous and endogenous ligands. Lipocalins share a conserved eight anti-parallel β-sheet structure. Among the different lipocalins identified in humans, α-1-acid glycoprotein (AGP), apolipoprotein D (apoD), apolipoprotein M (apoM), α1-microglobulin (α1-m) and retinol-binding protein (RBP) are plasma proteins. In particular, AGP is the most important transporter for basic and neutral drugs, apoD, apoM, and RBP mainly bind endogenous molecules such as progesterone, pregnenolone, bilirubin, sphingosine-1-phosphate, and retinol, while α1-m binds the heme. Human serum albumin (HSA) is a monomeric all-α protein that binds endogenous and exogenous molecules like fatty acids, heme, and acidic drugs. Changes in the plasmatic levels of lipocalins and HSA are responsible for the onset of pathological conditions associated with an altered drug transport and delivery. This, however, does not necessary result in potential adverse effects in patients because many drugs can bind both HSA and lipocalins, and therefore mutual compensatory binding mechanisms can be hypothesized. Here, molecular and clinical aspects of ligand transport by plasma lipocalins and HSA are reviewed, with special attention to their role as alterative carriers in health and disease.

Human serum albumin (HSA): (i) controls the plasma oncotic pressure, (ii) modulates fluid distribution between the body compartments, (iii) represents the depot and carrier of endogenous and exogenous compounds, (iv) increases the apparent solubility and lifetime of hydrophobic compounds, (v) affects pharmacokinetics of many drugs, (vi) inactivates toxic compounds, (vii) induces chemical modifications of some ligands, (viii) displays antioxidant properties, and (ix) shows enzymatic properties. Under physiological and pathological conditions, HSA has a pivotal role in heme scavenging transferring the metal-macrocycle from high- and low-density lipoproteins to hemopexin, thus acquiring globin-like reactivity. Here, the heme-based catalytic properties of HSA are reviewed and the structural bases of drug-dependent allosteric regulation are highlighted.

Retinoids are a class of chemicals derived from vitamin A metabolism, playing important and diverse functions. Vitamin A, also named all-trans-retinol (all-trans-ROL), is coverted into two classes of biologically active retinoids, i.e. 11-cis-retinoids and acidic retinoids. Among acidic retinoids, all-trans-retinoic acid (all-trans-RA) and 9-cis-retinoic acid (9-cis-RA) represent the main metabolic products. Specific and aspecific proteins solubilize, protect, and detoxify retinoids in the extracellular environment. The retinoid binding protein 4 (RBP4), the epididymal retinoid-binding protein (ERBP), and the inter photoreceptor matrix retinoid-binding protein (IRBP) play a central role in ROL transport, whereas lipocalin-type prostaglandin D synthase (also named beta-trace) and human serum albumin (HSA) transport preferentially all-trans-RA. Here, the modulatory effect of all-trans-RA and all-trans-ROL on ferric heme (heme-Fe(III)) binding to HSA is reported. All-trans-RA and all-trans-ROL binding to the FA1 site of HSA competitively inhibit heme-Fe(III) association. Docking simulations and local structural comparison of HSA with all-trans-RA- and all-trans-ROL-binding proteins support functional data indicating the preferential binding of all-trans-RA and all-trans-ROL to the FA1 site of HSA. Present results may be relevant in vivo, in fact HSA could act as a secondary carrier of retinoids in human diseases associated with reduced levels of RBP4 and IRBP.

The relationship between DNA repair failure and cancer is well established as in the case of rare, high penetrant genes in high cancer risk families. Beside this, in the last two decades, several studies have investigated a possible association between low penetrant polymorphic variants in genes devoted to DNA repair pathways and risk for developing cancer. This relationship would be also supported by the observation that DNA repair processes may be modulated by sequence variants in DNA repair genes, leading to susceptibility to environmental carcinogens. In this framework, the aim of this review is to provide the reader with the state of the art on the association between common genetic variants and cancer risk, limiting the attention to single nucleotide polymorphisms (SNPs) of the NBN gene and providing the various odd ratios (ORs). In this respect, the NBN protein, together with MRE11 and RAD50, is part of the MRN complex which is a central player in the very early steps of sensing and processing of DNA double-strand breaks (DSBs), in telomere maintenance, in cell cycle control, and in genomic integrity in general. So far, many papers were devoted to ascertain possible association between common synonymous and non-synonymous NBN gene polymorphisms and increased cancer risk. However, the results still remain inconsistent and inconclusive also in meta-analysis studies for the most investigated E185Q NBN miscoding variant.

Retinoic acid (RA), the major bioactive metabolite of retinol or vitamin A, induces a spectrum of pleiotropic effects in cell growth and differentiation that are relevant for embryonic development and adult physiology. The RA activity is mediated primarily by members of the retinoic acid receptor (RAR) subfamily, namely RARα, RARβ and RARγ, which belong to the nuclear receptor (NR) superfamily of transcription factors. RARs form heterodimers with members of the retinoid X receptor (RXR) subfamily and act as ligand-regulated transcription factors through binding specific RA response elements (RAREs) located in target genes promoters. RARs also have non-genomic effects and activate kinase signaling pathways, which fine-tune the transcription of the RA target genes. The disruption of RA signaling pathways is thought to underlie the etiology of a number of hematological and non-hematological malignancies, including leukemias, skin cancer, head/neck cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, liver cancer, glioblastoma and neuroblastoma. Of note, RA and its derivatives (retinoids) are employed as potential chemotherapeutic or chemopreventive agents because of their differentiation, anti-proliferative, pro-apoptotic, and anti-oxidant effects. In humans, retinoids reverse premalignant epithelial lesions, induce the differentiation of myeloid normal and leukemic cells, and prevent lung, liver, and breast cancer. Here, we provide an overview of the biochemical and molecular mechanisms that regulate the RA and retinoid signaling pathways. Moreover, mechanisms through which deregulation of RA signaling pathways ultimately impact on cancer are examined. Finally, the therapeutic effects of retinoids are reported.

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of K s, k +2, and k +2/K s obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k +3 << k +2). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pK a-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.

The cationic platinum complexes [Pt(η2-alkene)X(tmeda)](ClO4) (1: X = Cl, alkene = propene, 1-butene, 1-hexene; X = NO2, alkene = (E)-2-butene, (Z)-2-butene; tmeda = N,N,N‘,N‘-tetramethylethylenediamine) smoothly react with triethylamine to undergo loss of one allylic proton of the alkene ligand and formation of the η1-allyl species [Pt(η1-allyl)X(tmeda)] (2). In the case of 1-butene and 1-hexene, the η1-allyl species containing a nonterminal olefinic function (X(tmeda)PtCH2CHCHR, R = Me, Pr) undergoes release of the X ligand and η1 → η3 rearrangement of the allyl to form [Pt(η3-allyl)(tmeda)]+ (3). The reaction is stereoselective, and the R substituent is always found syn to the central hydrogen of the allyl group; however, in solution, a partial isomerization of the syn into the anti form slowly takes place. In the case of propene and (E)- and (Z)-2-butene the terminal olefinic function of 2, [X(tmeda)PtCHRCHCH2], can displace the η2-alkene from a second molecule of 1 to form the η1:η2-allyl-bridged di...

Truncated hemoglobins (trHbs) are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). Most trHbs are involved in the O2/NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host. Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mycobacterium tuberculosis trHbs N and O (Mt-trHbN and Mt-trHbO, respectively), and Campylobacter jejuni trHb (Cj-trHbP) nitrosylation, (ii) nitrite-mediated nitrosylation of ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP, and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO; however, the conversion of nitrite to NO is facilitated primarily by ferrous Mt-trHbN. Values of kinetic and/or thermodynamic parameters reflect specific trHb structural features, such as the ligand diffusion pathways to/from the heme, the heme distal pocket structure and polarity, and the ligand stabilization mechanisms. In particular, the high reactivity of Mt-trHbN and Cj-trHbP reflects the great ligand accessibility to the heme center by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the heme-Fe atom of Mt-trHbO the ligand accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo.

Human serum albumin (HSA), the most abundant protein in plasma, is a monomeric multi-domain macromolecule, representing the main determinant of plasma oncotic pressure and the main modulator of fluid distribution between body compartments. HSA displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds. Indeed, HSA represents the main carrier for fatty acids, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays (pseudo-)enzymatic properties. HSA is a valuable biomarker of many diseases, including cancer, rheumatoid arthritis, ischemia, post-menopausal obesity, severe acute graft-versus-host disease, and diseases that need monitoring of the glycemic control. Moreover, HSA is widely used clinically to treat several diseases, including hypovolemia, shock, burns, surgical blood loss, trauma, hemorrhage, cardiopulmonary bypass, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, nutrition support, resuscitation, and hypoalbuminemia. Recently, biotechnological applications of HSA, including implantable biomaterials, surgical adhesives and sealants, biochromatography, ligand trapping, and fusion proteins, have been reported. Here, genetic, biochemical, biomedical, and biotechnological aspects of HSA are reviewed.

The ibuprofen primary binding site FA3–FA4 is located in domain III of human serum albumin (HSA), the secondary clefts FA2 and FA6 being sited in domains I and II. Here, the thermodynamics of ibuprofen binding to recombinant Asp1–Glu382 truncated HSA (tHSA)–heme-Fe(III) and nitrosylated tHSA–heme-Fe(II), encompassing domains I and II only, is reported. Moreover, the allosteric effect of ibuprofen on the kinetics of tHSA–heme-Fe(III)-mediated peroxynitrite isomerization and nitrosylated tHSA–heme-Fe(II) denitrosylation has been investigated. The present data indicate, for the first time, that the allosteric modulation of tHSA–heme and HSA–heme reactivity by ibuprofen depends mainly on drug binding to the FA2 and FA6 secondary sites rather than drug association with the FA3–FA4 primary cleft. Thus, tHSA is a valuable model with which to investigate the allosteric linkage between the heme cleft FA1 and the ligand-binding pockets FA2 and FA6, all located in domains I and II of (t)HSA.

Two new cationic olefin complexes of platinum(II) with electron-withdrawing substituents on the allylic carbon, [PtCl(η2-CH2═CHCH2X)(tmeda)]+ (X = −C(O)OCH3 (1a), −OC(O)CH3 (1b)) have been synthesized and their reactivity toward nucleophiles investigated. The reaction of 1a with various nucleophiles, including water, causes allylic deprotonation and formation of the η3-allyl complex [Pt{η3-CH2CHCHC(O)OCH3}(tmeda)]+ (4a). In nonaqueous media it was possible to identify also a transient alkenyl species, [PtCl{η1-CH═CHCH2C(O)OCH3}(tmeda)] (2a), formed by loss of a vinylic proton. In contrast, the reaction of 1b with nucleophiles does not lead to deprotonation but to substitution of the acetate group followed by addition to the olefinic double bond. The latter can be intramolecular, as in the case of acetylacetonate, leading to [PtCl{η1-CH2CHCH2C[C(O)CH3]═C(CH3)O}(tmeda)] (5b) or requires a second molecule of nucleophile, as in the case of secondary amines, leading to the Markovnikov and anti-Markovnikov addi...

Heme endows human serum albumin (HSA) with heme-protein-like reactivity and spectroscopic properties. Here, the kinetics and thermodynamics of reductive nitrosylation of ferric human serum heme-albumin [HSA-heme-Fe(III)] are reported. All data were obtained at 20 °C. At pH 5.5, HSA-heme-Fe(III) binds nitrogen monoxide (NO) reversibly, leading to the formation of nitrosylated HSA-heme-Fe(III) [HSA-heme-Fe(III)-NO]. By contrast, at pH ≥ 6.5, the addition of NO to HSA-heme-Fe(III) leads to the transient formation of HSA-heme-Fe(III)-NO in equilibrium with HSA-heme-Fe(II)-NO+. Then, HSA-heme-Fe(II)-NO+ undergoes nucleophilic attack by OH− to yield ferrous human serum heme-albumin [HSA-heme-Fe(II)]. HSA-heme-Fe(II) further reacts with NO to give nitrosylated HSA-heme-Fe(II) [HSA-heme-Fe(II)-NO]. The rate-limiting step for reductive nitrosylation of HSA-heme-Fe(III) is represented by the OH−-mediated reduction of HSA-heme-Fe(II)-NO+ to HSA-heme-Fe(II). The value of the second-order rate constant for OH−-mediated reduction of HSA-heme-Fe(II)-NO+ to HSA-heme-Fe(II) is 4.4 × 103 m−1·s−1. The present results highlight the role of HSA-heme-Fe in scavenging reactive nitrogen species.

The reactivity of [PtCl(η2-CH2 CHR)(tmeda)]+ (R = H, 1a, or Me, 1b; tmeda = N,N,N′,N′-tetramethyl-1,2-diaminoethane) towards some ambident nucleophiles like anilines and phenolate anion has been tested. The reaction of 1a with N-methylaniline gives immediately N-addition to the coordinated ethene (3a), but, in the presence of an inorganic carbonate, a partial rearrangement, with the para carbon of the phenyl ring taking the place of nitrogen, is observed (4a and 5a). Reaction with a tertiary aromatic amine, such as N,N-dimethylaniline, leads exclusively to the C-coupled species. The phenolate anion acts initially as an oxygen donor, however the resulting species (6a), in contact with free phenol, rearranges to C-bonded species (7a). For free phenol/6a ratios ⩾ 5 the rearranged product has an isomeric ortho/para ratio of ≈3. For lower free phenol/6a ratios (⩽ 1) oligomeric complexes, in which two or three platinum ethanide moieties are bound to the same phenol ring, are also formed. In the case of 1b, the above described reactivity has to compete with the base-induced deprotonation of propene, leading to formation of the allyl-bridged platinum dimer [{PtCl(tmeda)}(μ-η1:η3-CHCHCH2){Pt(tmeda)}]+. The X-ray crystal structure of 1b has also been determined; the structural parameters are very similar to those previously reported for 1a. DFT calculations have shown a similar activation of the two complexes towards nucleophilic addition at the coordinated olefin, although in 1b the electrophilic character of the olefin is masked by the Bronsted acidity of the propene methyl protons.